Introduction of a new branchpoint in tetrapyrrole biosynthesis in Escherichia coli by co-expression of genes encoding the chlorophyll-specific enzymes magnesium chelatase and magnesium protoporphyrin methyltransferase

The genes encoding the three Mg chelatase subunits, ChlH, ChlI and ChlD, fr om the cyanobacteriurm Synechocystis PCC6803 were all cloned in the same pE T9a-based Escherichia coli expression plasmid, forming an artificial chlH-I -D operon under the control of the strong T7 promoter, When a soluble extra ct from IPTG-induced E, coli cells containing the pET9a-ChlHID plasmid was assayed for Mg chelatase activity in vitro, a high activity was obtained, s uggesting that all three subunits are present in a soluble and active form. The chlM gene of Synechocystis PCC6803 was also cloned in a pET-based E, c oil expression vector. Soluble extract from an E, coil strain expressing ch lM converted Mg-protoporphyrin IX to Mg-protoporphyrin monomethyl ester, de monstrating that chlM encodes the Mg-protoporphyrin methyltransferase of Sy nechocystis. Go-expression of the chlM gene together with the chlH-I-D cons truct yielded soluble protein extracts which converted protoporphyrin IX to Mg-protoporphyrin IX monomethyl ester without detectable accumulation of t he Mg-protoporphyrin IX intermediate, Thus, active Mg chelatase and Mg-prot oporphyrin IX methyltransferase can be coupled in E, coli extracts, Purifie d ChlI, -D and -H subunits in combination with purified ChlM protein were s ubsequently used to demonstrate in vitro that a molar ratio of ChlM to ChlH of 1 to 1 results in conversion of protoporphyrin IX to Mg-protoporphyrin monomethyl ester without significant accumulation of Mg-protoporphyrin, (C) 1999 Federation of European Biochemical Societies.